title: An improved targeted cAMP sensor to study the regulation of adenylyl cyclase 8 by Ca2+ entry through voltage-gated channels.
abstract: Here we describe an improved sensor with reduced pH sensitivity tethered to adenylyl cyclase (AC) 8. The sensor was used to study cAMP dynamics in the AC8 microdomain of MIN6 cells, a pancreatic β-cell line. In these cells, AC8 was activated by Ca(2+) entry through L-type voltage-gated channels following depolarisation. This activation could be reconstituted in HEK293 cells co-expressing AC8 and either the α1C or α1D subunit of L-type voltage-gated Ca(2+) channels. The development of this improved sensor opens the door to the study of cAMP microdomains in excitable cells that have previously been challenging due to the sensitivity of fluorescent proteins to pH changes.
authors.affiliation: Department of Pharmacology, University of Cambridge, Cambridge, United Kingdom.
authors.forename: Katy L
authors.initials: KL
authors.lastname: Everett
authors.forename: Dermot M F
authors.initials: DM
authors.lastname: Cooper
mesh_terms: D000262:Adenylyl Cyclases / Q000378:metabolism*; D001426:Bacterial Proteins / Q000378:metabolism; D015153:Blotting, Western; D002118:Calcium / Q000378:metabolism*; D015220:Calcium Channels / Q000378:metabolism*; D000242:Cyclic AMP / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D057809:HEK293 Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D050417:Insulin-Secreting Cells / Q000378:metabolism*; D008164:Luminescent Proteins / Q000378:metabolism; D018613:Microscopy, Confocal; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D001426:Bacterial Proteins; D015220:Calcium Channels; D017931:DNA Primers; D008164:Luminescent Proteins; C434479:citrine protein, bacteria; D000242:Cyclic AMP; D000262:Adenylyl Cyclases; C118292:adenylyl cyclase 8; D002118:Calcium